Cholesterol is a vital compound in maintenance for human health, and its concentration levels are tightly associated with various diseases. Therefore, accurate monitoring of cholesterol is of great significance in clinical diagnosis. Herein, we fabricated a noncontact biosensor based on photonic crystal enhanced upconversion nanoparticles (UCNPs) for highly sensitive and interference-free cholesterol detection. By compounding LiErF₄:0.5%Tm³⁺@LiYF₄UCNPs with poly(methyl- methacrylate) (PMMA) photonic crystals (OPCs), we were able to selectively tune the coupling of the photonic band gap to the excitation field and modulate the upconversion (UC) luminescence intensity, given the unique multi-wavelength excitation property of LiErF₄:0.5%Tm³⁺@LiYF₄. A 48.5-fold enhancement of the monochromatic red UC emission was ultimately achieved at 980 nm excitation, ensuring improved detection sensitivity. Based on the principle of quenching of the intense monochromic red UC emission by the oxidation products of 3,3′,5,5′- tetramethylbenzidine (TMB) yielded from the cholesterol cascade reactions, the biosensor has a detection limit of 1.6 μM for cholesterol with excellent specificity and stability. In addition, the testing results of the as-designed biosensor in patients are highly consistent with clinical diagnostic data, providing a sensitive, reliable, reusable, interference-free, and alternative strategy for clinical cholesterol detection.